Hindawi Publishing Corporation Oxidative Medicine and Cellular Longevity Volume 2014, Article ID 360438, 31 pages http://dx.doi.org/10.1155/2014/360438

Review Article Lipid Peroxidation: Production, Metabolism, and Signaling Mechanisms of Malondialdehyde and 4-Hydroxy-2-Nonenal

Antonio Ayala, Mario F. Muñoz, and Sandro Argüelles

Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Seville, Prof Garc´ıa Gonzales s/n., 41012 Seville, Spain

Correspondence should be addressed to Sandro Arguelles;¨ [email protected]

Received 14 February 2014; Accepted 24 March 2014; Published 8 May 2014

AcademicEditor:KotaV.Ramana

Copyright © 2014 Antonio Ayala et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Lipid peroxidation can be described generally as a process under which oxidants such as free radicals attack lipids containing carbon-carbon double bond(s), especially polyunsaturated fatty acids (PUFAs). Over the last four decades, an extensive body of literature regarding lipid peroxidation has shown its important role in cell biology and human health. Since the early 1970s, the total published research articles on the topic of lipid peroxidation was 98 (1970–1974) and has been increasing at almost 135-fold, by up to 13165 in last 4 years (2010–2013). New discoveries about the involvement in cellular physiology and pathology, as well as the control of lipid peroxidation, continue to emerge every day. Given the enormity of this field, this review focuses on biochemical concepts of lipid peroxidation, production, metabolism, and signaling mechanisms of two main omega-6 fatty acids lipid peroxidation products: malondialdehyde (MDA) and, in particular, 4-hydroxy-2-nonenal (4-HNE), summarizing not only its physiological and protective function as signaling molecule stimulating gene expression and cell survival, but also its cytotoxic role inhibiting gene expression and promoting cell death. Finally, overviews of in vivo mammalian model systems used to study the lipid peroxidation process, and common pathological processes linked to MDA and 4-HNE are shown.

This review paper is dedicated to Dr. Alberto Machado

1. Lipids Overview of Biological Functions and permeability. Lipids also have a key role in biology as signaling molecules.

Lipids Are Classically Divided into Two Groups: Apolar and Lipids as Signaling Molecules.Themainenzymesthatgenerate Polar. Triglycerides (apolar), stored in various cells, but lipid signaling mediators are lipoxygenase, which medi- especially in adipose (fat) tissue, are usually the main form of ate hydroperoxyeicosatetraenoic acids (HPETEs), lipoxins, energy storage in mammals [1, 2]. Polar lipids are structural leukotrienes, or hepoxilins biosynthesis after oxidation of components of cell membranes, where they participate in the arachidonic acid (AA) [4, 5], cyclooxygenase that produces formation of the permeability barrier of cells and subcellular prostaglandins [4], and cytochrome P-450 (CYP) which organelles in the form of a lipid bilayer. The major lipid type generates epoxyeicosatrienoic acids, leukotoxins, thrombox- defining this bilayer in almost all membranes is glycerol- ane, or prostacyclin [4]. Lipid signaling may occur via based phospholipid [3]. The importance of the membrane activation of a variety of receptors, including G protein- lipid physical (phase) state is evidenced by the fact that lipids coupled and nuclear receptors. Members of several different may control the physiological state of a membrane organelle lipid categories have been identified as potent intracellular by modifying its biophysical aspects, such as the polarity signal transduction molecules. Examples of signaling lipids 2 Oxidative Medicine and Cellular Longevity include (i) two derived from the phosphatidylinositol phos- ∙− Haber-Weiss reaction + O O2 phates, diacylglycerol (DAG) and inositol phosphates (IPs). H 2 DAG is a physiological activator of protein kinase C [6, 7] ∙ (n+1) n + HO 2 M M and transcription factor nuclear factor-kB (NF-𝜅B), which H promotes cell survival and proliferation. Diacylglycerol also ∙ H2O2 OH + H2O interacts indirectly with other signalling molecules such as Fenton reaction small G proteins [8]. IPs are a highly charged family of Figure 1: Fenton and Haber-Weiss reaction. Reduced form of lipid-derived metabolites, involved in signal transduction 𝑛 transition-metals (M ) reacts trough the Fenton reaction with that results in activation of Akt, mTOR [9], and calcium- ∙ hydrogen peroxide (H2O2), leading to the generation of OH. homeostasis [10, 11]; (ii) sphingosine-1-phosphate, a sph- ∙− Superoxide radical (O2 ) can also react with oxidized form of ingolipid derived from ceramide that is a potent messen- (𝑛+1) transition metals (M ) in the Haber-Weiss reaction leading to the 𝑛 ger molecule involved in regulating calcium mobilization, production of M , which then again affects redox cycling. migration, adhesion, and proliferation [12–14]; (iii) the prostaglandins, which are one type of fatty-acid derived eicosanoid involved in inflammation15 [ , 16]andimmunity 3+ [17]; (iv) phosphatidylserine, a phospholipid that plays an when superoxide reacts with ferric iron (Fe ). In addition important role in a number of signaling pathways, includes totheironredoxcyclingdescribedabove,alsoanumberof other transition-metal including Cu, Ni, Co, and V can be kinases, small GTPases, and fusogenic proteins [18]; (v) the ∙ responsible for HO formation in living cells (Figure 1). steroid hormones such as estrogen, testosterone, and cortisol, ∙ which modulate a host of functions such as reproduction, The hydroperoxyl radical (HO 2) plays an important metabolism, stress response, inflammation, blood pressure, role in the chemistry of lipid peroxidation. This protonated and salt and water balance [19]. form of superoxide yields H2O2 which can react with redox active metals including iron or copper to further generate ∙ ∙ HO through Fenton or Haber-Weiss reactions. The HO 2 2. Lipids Damage by Reactive Oxygen Species is a much stronger oxidant than superoxide anion-radical and could initiate the chain oxidation of polyunsaturated One of the consequences of uncontrolled oxidative stress phospholipids, thus leading to impairment of membrane (imbalance between the prooxidant and antioxidant levels function [28–30]. in favor of prooxidants) is cells, tissues, and organs injury caused by oxidative damage. It has long been recognized that 2.1. Lipid Peroxidation Process. Lipid peroxidation can be high levels of free radicals or reactive oxygen species (ROS) described generally as a process under which oxidants such can inflict direct damage to lipids. The primary sources of as free radicals or nonradical species attack lipids containing endogenous ROS production are the mitochondria, plasma carbon-carbondoublebond(s),especiallypolyunsaturated membrane, endoplasmic reticulum, and peroxisomes [20] fatty acids (PUFAs) that involve hydrogen abstraction from through a variety of mechanisms including enzymatic reac- a carbon, with oxygen insertion resulting in lipid per- tions and/or autooxidation of several compounds, such oxyl radicals and hydroperoxides as described previously as catecholamines and hydroquinone. Different exogenous [31]. Glycolipids, phospholipids (PLs), and cholesterol (Ch) stimuli, such as the ionizing radiation, ultraviolet rays, are also well-known targets of damaging and potentially tobacco smoke, pathogen infections, environmental toxins, lethal peroxidative modification. Lipids also can be oxi- and exposure to herbicide/insecticides, are sources of in vivo dized by enzymes like lipoxygenases, cyclooxygenases, and ROS production. cytochrome P450 (see above, lipid as signaling molecules). The two most prevalent ROS that can affect profoundly ∙ In response to membrane lipid peroxidation, and accord- the lipids are mainly hydroxyl radical (HO ) and hydroper- ∙ ∙ ing to specific cellular metabolic circumstances and repair oxyl (HO 2). The hydroxyl radical (HO )isasmall,highly capacities, the cells may promote cell survival or induce mobile, water-soluble, and chemically most reactive species cell death. Under physiological or low lipid peroxidation ofactivatedoxygen.Thisshort-livedmoleculecanbepro- rates (subtoxic conditions), the cells stimulate their mainte- duced from O2 in cell metabolism and under a variety of nance and survival through constitutive antioxidants defense stress conditions. A cell produces around 50 hydroxyl radicals systems or signaling pathways activation that upregulate every second. In a full day, each cell would generate 4 antioxidants proteins resulting in an adaptive stress response. million hydroxyl radicals, which can be neutralized or attack By contrast, under medium or high lipid peroxidation rates biomolecules [21]. Hydroxyl radicals cause oxidative damage (toxic conditions) the extent of oxidative damage overwhelms to cells because they unspecifically attack biomolecules22 [ ] repair capacity, and the cells induce apoptosis or necrosis located less than a few nanometres from its site of generation programmed cell death; both processes eventually lead to and are involved in cellular disorders such as neurodegenera- molecularcelldamagewhichmayfacilitatedevelopmentof tion [23, 24], cardiovascular disease [25], and cancer [26, 27]. ∙ various pathological states and accelerated aging. The impact It is generally assumed that HO in biological systems is of lipids oxidation in cell membrane and how these oxidative formed through redox cycling by Fenton reaction, where free 2+ damages are involved in both physiological processes and iron (Fe ) reacts with hydrogen peroxide (H2O2)andthe major pathological conditions have been analysed in several 2+ Haber-Weiss reaction that results in the production of Fe reviews [32–35]. Oxidative Medicine and Cellular Longevity 3

The overall process of lipid peroxidation consists of three free radicals.”In particular 4-HNE, which has been subjected steps: initiation, propagation, and termination [31, 36, 37]. to intense scientific scrutiny in 90s [49], is considered In the lipid peroxidation initiation step, prooxidants like as “one of the major toxic products generated from lipid hydroxyl radical abstract the allylic hydrogen forming the peroxides” [49]. 4-HNE high toxicity can be explained by its ∙ carbon-centered lipid radical (L ). In the propagation phase, rapid reactions with thiols and amino groups [56]. Reactive ∙ lipid radical (L ) rapidly reacts with oxygen to form a lipid aldehydes, especially 4-HNE, act both as signaling molecules ∙ peroxy radical (LOO ) which abstracts a hydrogen from (see below 4-HNE as signaling molecule) and as cytotoxic ∙ another lipid molecule generating a new L (that continues products of lipid peroxidation causing long-lasting biological the chain reaction) and lipid hydroperoxide (LOOH). In the consequences, in particular by covalent modification of termination reaction, antioxidants like vitamin E donate a macromolecules (seebelow4-HNEbiomolecularadducts). ∙ hydrogen atom to the LOO species and form a correspond- 4-HNE is considered as “second toxic messengers of free ∙ ing vitamin E radical that reacts with another LOO forming radicals,” and also as “one of the most physiologically active nonradical products (Figure 2). Once lipid peroxidation is lipid peroxides,” “one of major generators of oxidative stress,” initiated, a propagation of chain reactions will take place until “a chemotactic aldehydic end-product of lipid peroxidation,” termination products are produced. Review with extensive and a “major lipid peroxidation product” [57]. Thus, it information regarding the chemistry associated with each of is not a surprise that 4-HNE is nowadays considered as thesestepsisavailable[31]. major bioactive marker of lipid peroxidation and a signaling molecule involved in regulation of several transcription factors sensible to stress such as nuclear factor erythroid 2.2. Lipid Peroxidation Products. Lipid peroxidation or reac- 2-related factor 2 (Nrf2), activating protein-1 (AP-1), NF- tion of oxygen with unsaturated lipids produces a wide 𝜅B, and peroxisome-proliferator-activated receptors (PPAR), variety of oxidation products. The main primary products in cell proliferation and/or differentiation, cell survival, of lipid peroxidation are lipid hydroperoxides (LOOH). autophagy, senescence, apoptosis, and necrosis (see below 4- Among the many different aldehydes which can be formed as HNE as signaling molecule). secondary products during lipid peroxidation, malondialde- Characteristics of various lipid peroxidation products as hyde (MDA), propanal, hexanal, and 4-hydroxynonenal (4- biomarkers have been reviewed on the basis of mechanisms HNE) have been extensively studied by Esterbauer and his and dynamics of their formation and metabolism and also colleagues in the 80s [38–49]. MDA appears to be the most on the methods of measurement, with an emphasis on the mutagenic product of lipid peroxidation, whereas 4-HNE is advantages and limitations [58]. the most toxic [50]. MDA has been widely used for many years as a convenient biomarker for lipid peroxidation of omega-3 and omega-6 2.3. Primary Lipid Peroxidation Product-Lipid Hydroperox- fatty acids because of its facile reaction with thiobarbituric ides. Hydroperoxides are produced during the propagation acid (TBA) [48, 51]. The TBA test is predicated upon the phase constituting the major primary product of lipid perox- reactivity of TBA toward MDA to yield an intensely colored idation process. The hydroperoxide group may be attached chromogen fluorescent red adduct; this test was first used by to various lipid structures, for example, free fatty acids, tria- food chemists to evaluate autoxidative degradation of fats and cylglycerols, phospholipids, and sterols. Lipid hydroperoxide oils [52].However,thethiobarbituricacidreactingsubstances generation, turnover and effector action in biological systems test (TBARS) is notoriously nonspecific which has led to have been reviewed [36]. In contrast to free radical, usually substantial controversy over its use for quantification of MDA highly reactive and chemically unstable, at moderate reaction from in vivo samples. Several technologies for the determi- conditions, such as low temperature and absence of metal nation of free and total MDA, such gas chromatography- ions, lipid hydroperoxides are relatively more stable products. mass spectrometry (GC-MS/MS), liquid chromatography- We found that lipid hydroperoxides in serum could be useful mass spectrometry (LC-MS/MS), and several derivatization- to predict the oxidative stress in tissues [59], and the levels based strategies, have been developed during the last decade of oxidative stress, including lipid peroxidation, increased [53]. Because MDA is one of the most popular and reliable throughout the day [60]. Once formed lipid hydroperoxides markers that determine oxidative stress in clinical situations canbetargetofdifferentreductionreactions,resulting [53], and due to MDA’s high reactivity and toxicity underlying in peroxidative damage inhibition or peroxidative damage thefactthatthismoleculeisveryrelevanttobiomedical induction. research community. 4-HNE was first discovered in 60s [54]. Later, in 80s Peroxidative Damage Inhibition. Hydroperoxides may 4-HNE was reported as a cytotoxic product originating decompose in vivo through two-electron reduction, which from the peroxidation of liver microsomal lipids [40]. 4- caninhibittheperoxidativedamage.Theenzymesmainly Hydroxyalkenals produced in the course of biomembrane responsible for two-electron reduction of hydroperoxides lipids peroxidation, elicited either by free radicals or by are selenium-dependent glutathione peroxidases (GPx) chemicals, might exert a genotoxic effect in humans55 [ ]. The and selenoprotein P (SeP). GPxs are known to catalyze the 4-hydroxyalkenals are the most significant products because reduction of H2O2 or organic hydroperoxides to water or they are produced in relatively large amounts, and they are the corresponding alcohols, respectively, typically using very reactive aldehydes that act as “second messengers of glutathione (GSH) as reductant. Widely distributed in 4 Oxidative Medicine and Cellular Longevity

Antioxidant ∙ 4 OO

O2 Lipid peroxyl radical Unsaturated lipid 2 3 OOH

Rearrangement Lipid hydroperoxide

Unsaturated lipid radical ∙ + R H 1

Unsaturated lipid

Figure 2: Lipid peroxidation process. In Initiation, prooxidants abstract the allylic hydrogen forming the carbon-centered lipid radical; the carbon radical tends to be stabilized by a molecular rearrangement to form a conjugated diene (step 1). In the propagation phase, lipid radical rapidly reacts with oxygen to form a lipid peroxy radical (step 2) which abstracts a hydrogen from another lipid molecule generating a new lipid radical and lipid hydroperoxide (step 3). In the termination reaction, antioxidants donate a hydrogen atom to the lipid peroxy radical species resulting in the formation of nonradical products (step 4).

mammalian tissues GPx can be found in the cytosol, nuclei, acid (a high reactive species produced enzymatically by and mitochondria [61, 62]. The presence of selenocysteine myeloperoxidase [65, 66], which utilizes hydrogen peroxide (in the catalytic centre of glutathione peroxidases) as the to convert chloride to hypochlorous acid at sites of inflam- catalytic moiety was suggested to guarantee a fast reaction mation) yielding singlet molecular oxygen [67, 68]. Singlet 1 with the hydroperoxide and a fast reducibility by GSH oxygen (molecular oxygen in its first excited singlet state Δ 𝑔; [61].SePisthemajorselenoproteininhumanplasmathat 1 1 O2) can react with amino acid, and proteins resulting in reduced phospholipid hydroperoxide using glutathione or multiple effects including oxidation of side-chains, backbone thioredoxin as cosubstrate. It protected plasma proteins fragmentation, dimerization/aggregation, unfolding or con- against peroxynitrite-induced oxidation and nitration or formational changes, enzymatic inactivation, and alterations low-density-lipoproteins (LDL) from peroxidation [62]. in cellular handling and turnover of proteins [69, 70]. Major substrates for lipid peroxidation are polyunsatu- Peroxidative Damage Induction. Hydroperoxides may also rated fatty acids (PUFAs) [31, 36, 37], which are a family decompose in vivo through one-electron reduction and take of lipids with two or more double bounds, that can be part in initiation/propagation steps [31, 36, 37], induce new classified in omega-3 (n-3) and omega-6 (n-6) fatty acids lipid hydroperoxides, and feed the lipid peroxidation process; accordingtothelocationofthelastdoublebondrelativeto all these mechanisms can contribute to peroxidative damage the terminal methyl end of the molecule. The predominant n- induction/expansion. Lipid hydroperoxides can be converted 6 fatty acid is arachidonic acid (AA), which can be reduced (i) to oxygen radicals intermediates such as lipid peroxyl radical ∙ ∙ via enzymatic peroxidation to prostaglandins, leukotrienes, (LOO ) and/or alkoxyl (LO ) by redox cycling of transition thromboxanes, and other cyclooxygenase, lipoxygenase or metal (M), resulting in lipid hydroperoxide decomposition cytochrome P-450 derived products [4]; or (ii) via nonen- and the oxidized or reduced form of theses metal, respectively zymatic peroxidation to MDA, 4-HNE, isoprostanes, and [63]. The lipid peroxyl and alkoxyl radicals can attack other other lipid peroxidation end-products (more stables and toxic lipids promoting the propagation of lipid peroxidation than hydroperoxides) through oxygen radical-dependent 𝑛 ∙ − 𝑛+1 LOOH + M 󳨀→ LO + OH + M (1) oxidative routes [49, 71]. The continued oxidation of fatty acid side-chains and released PUFAs, and the fragmentation 𝑛+1 ∙ + 𝑛 LOOH + M 󳨀→ LOO + H + M . (2) of peroxides to produce aldehydes, eventually lead to loss of membrane integrity by alteration of its fluidity which Lipid hydroperoxides can also react with peroxynitrite (a finally triggers inactivation of membrane-bound proteins. short-lived oxidant species that is a potent inducer of cell Contrary to radicals that attack biomolecules located less death [64] and is generated in cells or tissues by the reaction than a few nanometres from its site of generation [22], the of nitric oxide with superoxide radical) or hypochlorous lipid peroxidation-derived aldehydes can easily diffuse across Oxidative Medicine and Cellular Longevity 5 membranes and can covalently modify any protein in the Through nonenzymatic oxygen radical-dependent reaction, cytoplasm and nucleus, far from their site of origin [72]. AA is the main precursor of bicyclic endoperoxide, which then undergoes further reactions with or without the partic- 2.4. Secondary Lipid Peroxidation Products: MDA. MDA is an ipationofothercompoundstoformMDA(Figure 3)[31, 49, end-product generated by decomposition of arachidonic acid 94, 95]. However, it should be possible that other eicosanoids and larger PUFAs [49], through enzymatic or nonenzymatic that can also be generated by nonenzymatic oxygen radical- processes (Figure 3). MDA production by enzymatic pro- dependent reaction [96–99] may be precursor of bicyclic cesses is well known but its biological functions and its possi- endoperoxide and MDA. Recent review has addressed the ble dose-dependent dual role have not been studied although pathways for the nonenzymatic formation of MDA under MDA is more chemically stable and membrane-permeable specific conditions [100]. than ROS and less toxic than 4-HNE and methylglyoxal (MG) [49].Sofar,onlyfewpapershavereportedthatMDAmay MDA Metabolism. Once formed MDA can be enzymatically act as signaling messenger and regulating gene expression: metabolizedorcanreactoncellularandtissularproteinsor (i) very recent research indicated that MDA acted as a DNA to form adducts resulting in biomolecular damages. signaling messenger and regulated islet glucose-stimulated Early studies showed that a probable biochemical route insulin secretion (GSIS) mainly through Wnt pathway. The for MDA metabolism involves its oxidation by mitochon- moderatelyhighMDAlevels(5and10𝜇M) promoted islet drial aldehyde dehydrogenase followed by decarboxylation 2+ GSIS, elevated ATP/ADP ratio and cytosolic Ca level, and to produce acetaldehyde, which is oxidized by aldehyde affected the gene expression and protein/activity production dehydrogenase to acetate and further to CO2 and H2O of the key regulators of GSIS [73]; (ii) in hepatic stellate cells, (Figure 3)[49, 101, 102]. On the other hand, phosphoglucose MDA induced collagen-gene expression by upregulating isomerase is probably responsible for metabolizing cytoplas- specificity protein-1Sp1 ( ) gene expression and Sp1 and Sp3 mic MDA to methylglyoxal (MG) and further to D-lactate protein levels [74]. Both Sp1 and Sp3 can interact with and by enzymes of the glyoxalase system by using GSH as a recruit a large number of proteins including the transcrip- cofactor [103]. A portion of MDA is excreted in the urine as tion initiation complex, histone modifying enzymes, and various enaminals (RNH-CH–CH-CHO) such as N-epsilon- chromatin remodeling complexes, which strongly suggest (2-propenal)lysine, or N-2-(propenal) serine [49]. that Sp1 and Sp3 are important transcription factors in the remodeling chromatin and the regulation of gene expression [75]. On the other hand, MDA production by nonenzymatic 2.4.1. MDA Biomolecules Adducts. As a bifunctional elec- processes remains poorly understood despite their potential trophile aldehyde, MDA reactivity is pH-dependent, which therapeutic value, because this MDA is believed to originate exists as enolate ion (conjugate bases having a negative under stress conditions and has high capability of reaction charge on oxygen with adjacent C–C double bond) with low with multiple biomolecules such as proteins or DNA that reactivity at physiological pH. When pH decreases MDA leadstotheformationofadducts[76–78], and excessive MDA exists as beta-hydroxyacrolein and its reactivity increases production have been associated with different pathological [49]. MDA’s high reactivity is mainly based on its elec- states [79–85](seeTable 1). Identifying in vivo MDA produc- trophilicity making it strongly reactive toward nucleophiles, tion and its role in biology is important as indicated by the such as basic amino acid residues (i.e., lysine, histidine, or extensive literature on the compound (over 15 800 articles in arginine). Initial reactions between MDA and free amino the PubMed database using the keyword “malondialdehyde acids or protein generate Schiff-base adducts49 [ , 104, 175]. lipid peroxidation” in December 2013). These adducts are also referred to as advanced lipid per- oxidation end-products (ALEs). Acetaldehyde (product of MDA Production by Enzymatic Processes.MDAcanbe MDA metabolism) under oxidative stress and in the presence generated in vivo as a side product by enzymatic processes of MDA further generates malondialdehyde acetaldehyde during the biosynthesis of thromboxane A2 (Figure 3)[86– (MAA) adducts [157, 176]. MAA adducts are shown to be 90]. TXA2 is a biologically active metabolite of arachidonic highly immunogenic [177–181]. MDA adducts are biologically acid formed by the action of the thromboxane A2 synthase, important because they can participate in secondary delete- on prostaglandin endoperoxide or prostaglandin H2 (PGH2) rious reactions (e.g., crosslinking) by promoting intramolec- [4, 91, 92]. PGH2 previously is generated by the actions of ular or intermolecular protein/DNA crosslinking that may cyclooxygenases on AA [4, 91, 93]. induce profound alteration in the biochemical properties of biomoleculesandaccumulateduringagingandinchronic MDA Production by Nonenzymatic Processes.Amixtureof diseases [72, 104, 182, 183].Importantproteinsthatcanbe lipid hydroperoxides is formed during lipid peroxidation modified by MDA adducts are as follows: (i) eElongation process. The peroxyl radical of the hydroperoxides with a factor 2 (eEF2) catalyzes the movement of the ribosome along cis-double bond homoallylic to the peroxyl group permits the mRNA in protein synthesis. MDA adducts with eEF2 their facile cyclization by intramolecular radical addition to could contribute to decline of protein synthesis, secondary to the double bond and the formation of a new radical. The LP increase (see below—cumene hydroperoxide-induced lipid intermediate free radicals formed after cyclization can cyclize peroxidation); (ii) factor H (FH) is the main regulator of the again to form bicycle endoperoxides, structurally related alternative pathway in plasma that tightly controls the activa- to prostaglandins, and undergo cleavage to produce MDA. tion of complement to prevent attack against host cells. MDA 6 Oxidative Medicine and Cellular Longevity

Table 1: Common pathological processes linked to MDA and 4-HNE.

Pathological processes Aldehyde References Alzheimer’s disease MDA [104–113] 4-HNE [81, 108, 114–121] Cancer MDA [109, 122–130] 4-HNE [72, 126–128, 131–136] Cardiovascular diseases MDA [72, 79, 109, 123, 135, 137–141] 4-HNE [72, 104, 109, 131, 135, 138, 139, 142–144] Diabetes MDA [79, 109, 123, 140, 145–150] 4-HNE [131, 135, 142, 143, 151–156] Liver disease MDA [123, 135, 157–164] 4-HNE [135, 160–163, 165–169] Parkinson’s disease MDA [81, 108, 114–121] 4-HNE [72, 114, 131, 135, 142, 170–174]

∙ Radical 2 PUFA O2 + H AA

∙ PUFA-radical 1

O2

+ H PUFA peroxide- PGG2 ∙ Cyclization radical 2

Oxy radical Lipid hydroperoxide + O2 + H PGH2

Bicyclic 3 3 3 endoperoxide

Monocyclic TXA2 HHT peroxide MDA

4 MDA-protein adducts MDA-DNA adducts

CO2 + H2O Malonic semialdehyde

7 5 Biomolecular damage cell death 6 4 Acetyl Acetate Acetaldehyde CoA

Figure 3: MDA formation and metabolism. MDA can be generated in vivo by decomposition of arachidonic acid (AA) and larger PUFAs as a side product by enzymatic processes during the biosynthesis of thromboxane A2(TXA2) and 12-l-hydroxy-5,8,10-heptadecatrienoic acid (HHT) (blue pathway), or through nonenzymatic processes by bicyclic endoperoxides produced during lipid peroxidation (red pathway). One formed MDA can be enzymatically metabolized (green pathway). Key enzymes involved in the formation and metabolism of MDA: cyclooxygenases (1), prostacyclin hydroperoxidase (2), thromboxane synthase (3), aldehyde dehydrogenase (4), decarboxylase (5), acetyl CoAsynthase(6),andtricarboxylicacidcycle(7). Oxidative Medicine and Cellular Longevity 7 adductswithFHcanblockboththeuptakeofMDA-modified acetal or can be target to Schiff-base formation, oxidation, or proteins by macrophages and MDA-induced proinflamma- reduction, and (iii) hydroxyl group which can be oxidized to tory effects in vivo in mice [184]; MDA adducts or MAA aketone[56]. adducts can promote binding of complement; (iii) anaphyla- 4-HNE is the most intensively studied lipid peroxidation toxin C3a (proinflammatory complement components) with end-product, in relation not only to its physiological and oxidatively modified low-density lipoproteins (Ox-LDL) and protective function as signaling molecule stimulating gene contributes to inflammatory processes involving activation expression, but also to its cytotoxic role inhibiting gene of the complement system in atherosclerosis [185]; and (iv) expression and promoting the development and progression protein kinase C (PKC) is known to play a major role in of different pathological states. In the last three years, intracellular signal transduction affecting such processes as excellent reviews have been published summarizing both proliferation, differentiation, migration, inflammation, and signaling and cytotoxic effects of this molecule in biology, for cytoskeletal organization. BSA-MAA induces the activation example, overview of mechanisms of 4-HNE formation and of a specific isoform of PKC, PKC-𝛼,inhepaticstellatecells most common methods for detecting and analyzing 4-HNE (HSCs)andinducestheincreasedsecretionofurokinase- anditsproteinadducts[196]. Review focuses on membrane type plasminogen activator, a key component of the plasmin- proteins affected by lipid peroxidation-derived aldehydes, generating system, thereby contributing to the progression under physiological and pathological conditions [131]. of hepatic fibrosis [186].Arecentreviewshowsalistofup Jaganjac and Co-workers have described the role of 4-HNE as to thirty-three proteins known to be modified by MDA and second messengers of free radicals that act both as signaling including enzymatic proteins, carrier proteins, cytoskeletal molecules and as cytotoxic products of lipid peroxidation proteins, and mitochondrial and antioxidant proteins [76]. involvement in the pathogenesis of diabetes mellitus (DM) It has also been proposed that MDA could react phys- [151]. Chapple and Co-workers summarized the production, iologically with several nucleosides (deoxy-guanosine and metabolism and consequences of 4-HNE synthesis within cytidine) to form adducts to deoxyguanosine and deoxya- vascular endothelial, smooth muscle cells and targeted denosine, and the major product resulting is a pyrimidop- signaling within vasculature [142]. Review focuses on the role urinone called pyrimido[1,2-a]purin-10(3H-)one (M1G or of 4-HNE and Ox-PLs affecting cell signaling pathways and M1dG) [122, 123, 187, 188]. MDA is an important contributor endothelial barrier dysfunction through modulation of the to DNA damage and mutation [122, 124]. The main route for activities of proteins/enzymes by Michael adducts formation, repair of M1dG residues in genomic DNA appears to be the enhancing the level of protein tyrosine phosphorylation of nucleotide excision repair (NER) pathway [188, 189]. In the the target proteins, and by reorganization of cytoskeletal, absence of repair, MDA-DNA adducts may lead to mutations focal adhesion, and adherens junction proteins [197]. An (point and frameshift)124 [ ], strand breaks [122, 190], cell overview of molecular mechanisms responsible for the cycle arrest [191], and induction of apoptosis [192]. M1dG is overall chemopreventive effects of sulforaphane (SFN), oxidized to 6-oxo-M1dG in rats and that xanthine oxidase focusing on the role of 4-HNE in these mechanisms, (XO) and aldehyde oxidase (AO) are the likely enzymes which may also contribute to its selective cytotoxicity to responsible [193]. This MDA-induced DNA alteration may cancer cells [198]. Perluigi and Co-workers summarized the contribute significantly to cancer and other genetic diseases. role of lipid peroxidation, particularly of 4-HNE-induced Hypermethylatedincancer1(HIC1)isatumorsuppressor protein modification, in neurodegenerative diseases. gene that cooperates with p53 to suppress cancer develop- In this review, the authors also discuss the hypothesis ment. New funding has shown that highest HIC1 methylation that altered energy metabolism, reduced antioxidant levels in tobacco smokers were significantly correlated with defense, and mitochondrial dysfunction are characteristic oxidative DNA adducts M1dG [125]. Research also suggests hallmarks of neurodegenerative [170]. Zimniak described that persistent M1dG adducts in mitochondrial DNA hinder the effects of 4-HNE and other endogenous electrophiles on the transcription of mitochondrial genes [194]. Dietary intake longevity, and its possible molecular mechanisms. The role of certain antioxidants such as vitamins was associated with of electrophiles is discussed, both as destabilizing factors reduced levels of markers of DNA oxidation (M1dG and 8- and as signals that induce protective responses [199]. Reed oxodG) measured in peripheral white blood cells of healthy showed the relationship between lipid peroxidation/4- subjects, which could contribute to the protective role of HNE and neurodegenerative diseases. It also demonstrates vitamins on cancer risk [195]. how findings in current research support the common themes of altered energy metabolism and mitochondrial dysfunction in neurodegenerative disorders [171]. Fritz 2.5. Secondary Lipid Peroxidation Products: 4-HNE. 4- and Petersen summarized the generation of reactive Hydroxynonenal (4-HNE), 𝛼, 𝛽-unsaturated electrophilic aldehydes via lipid peroxidation resulting in protein compounds, is the major type of 4-hydroxyalkenals end- carbonylation, and pathophysiologic factors associated with product, generated by decomposition of arachidonic acid and 4-HNE-protein modification. Additionally, an overview larger PUFAs, through enzymatic or nonenzymatic processes of in vitro and in vivo model systems used to study the [49]. 4-HNE is an extraordinarily reactive compound con- physiologic impact of protein carbonylation, and an update taining three functional groups: (i) C=C double bond that of the methods commonly used in characterizing protein can be target to Michael additions to thiol, reduction or modification by reactive aldehydes [200]. Butterfield and Co- epoxidation, (ii) carbonyl group which can yield acetal/thio workers showed that several important irreversible protein 8 Oxidative Medicine and Cellular Longevity modifications including protein nitration and 4-HNE Alkenal derived modification, both which have been extensively investigated HP-Lyase Alkenal OX in research on the progression of Alzheimer’s disease (AD) [201]. Balogh and Atkins described the cellular effects of 4-HNE, followed by a review of its GST-catalyzed 9-HPODE 4-HPNE detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha- class GSTs. Additionally, a summary of the literature that 15-LOX Peroxygenase examines the interplay between GSTs and 4-HNE in model LA 4-HNE systems relevant to oxidative stress is also discussed to demonstrate the magnitude of importance of GSTs in the ADH overall detoxification scheme202 [ ]. Like MDA, 4-HNE has GSH

high capability of reaction with multiple biomolecules such ALDH as proteins or DNA that lead to the formation of adducts ADH [49]. GH-DHN GS-HNE HNA DHN

4-HNE Production by Enzymatic Processes. 4-HNE is a CYP lipid peroxidation end-product of enzymatic transforma- ALDH tion of n-6 PUFAs (AA, linoleic acid, and other) by 15- GH-HNA 9-OH-HNA lipoxygenases (15-LOX). Two different 15-LOX exist, (i) 15-LOX-1 (reticulocyte type) expressed in reticulocytes, Figure 4: Enzymatic production of 4-HNE and metabolism. In eosinophils, and macrophages; (ii) and 15-LOX-2 (epidermis plant enzymatic route to 4-HNE includes lipoxygenase (LOX), type) expressed in skin, cornea, prostate, lung, and esophagus -hydroperoxide lyase (HPL), alkenal oxygenase (AKO), and per- [203–205]. Mice do not express 15-LOX and only express oxygenases. 4-HNE metabolism may lead to the formation of the leukocyte-derived 12-LOX. In plant enzymatic route corresponding alcohol 1,4-dihydroxy-2-nonene (DHN), corre- to 4-HNE includes lipoxygenase (LOX), -hydroperoxide sponding acid 4-hydroxy-2-nonenoic acid (HNA), and HNE– lyase (HPL), alkenal oxygenase (AKO), and peroxygenases glutathione conjugate products. 4-HNE conjugation with glu- tathione s-transferase (GSH) produce glutathionyl-HNE (GS-HNE) (Figure 4)[206]. The main precursors of 4-HNE in human followed by NADH-dependent alcohol dehydrogenase (ADH- are 13-hydroperoxyoctadecadienoic acid (13-HPODE) pro- )catalysed reduction to glutathionyl-DNH (GS-DNH) and/or alde- duced by the oxidation of linoleic acid by 15-LOX-1 [207]and hyde dehydrogenase (ALDH-)catalysed oxidation to glutathionyl- 15- hydroperoxyeicosatetraenoic acids (15-HPETE) produced HNA (GS-HNA). 4-HNE is metabolized by ALDH yielding HNA, by the oxidation of AA by 15-LOX-2 [208]. These compounds which is metabolized by cytochrome P450 (CYP) to form 9- areshortlivedandarecatabolisedintovariousfamilies hydroxy-HNA (9-OH-HNA). 4-HNE may be also metabolized by of more stable compounds such as 15-HETEs, lipoxins, ADH to produce DNH. and leukotrienes [4]. 15-HPETE is associated with anti- inflammatory and proapoptotic functions (the release of cytochrome c, activation of caspase-3 and 8, PARP, and Bid of their structure produce another radical intermediate that cleavage) and DNA fragmentation [209, 210]. after oxygenation step forms the corresponding dihydroper- oxyde derivative (unstable), which after Hock rearrange- 4-HNEProductionbyNonenzymaticProcesses. 4-HNE can ment and cleavage produces 4-hydroperoxy-2E-nonenal (4S- be formed through several nonenzymatic oxygen radical- HPNE), immediate precursor of HNE; and (v) the oxida- dependent routes involving the formation of hydroperoxides, tion products generated after reaction of linoleate-derived +2 alkoxyl radicals, epoxides, and fatty acyl crosslinking reac- hydroperoxy epoxide (13-Hp-Epo-Acid) with Fe yields an tions. Spickett C [196]recentlyreviewedthemechanismsof alkolxyl radical, which undergo to di-epoxy-carbinyl radical formation of 4-HNE during lipid peroxidation and showed and after beta-scission yield different aldehydes compounds that the main processes leading to 4-HNE are likely beta- including 4-HNE (Figure 5). cleavage reaction of lipid alkoxy-radicals, which can be Once formed 4-HNE, and depending of cell type and summarized into five generic mechanisms: (i) reduction of cellular metabolic circumstances can promote cell survival or the hydroperoxide to a lipid alkoxy radical by transition metal 2+ death. Cells expressing differentiated functions representative ions, such as Fe followed by b-scission; (ii) protonation of for the in vivo situation react more sensitively to 4-HNE than the lipid hydroperoxide yields an acidified lipid hydroperox- cell lines. The different response with respect to the endpoints ide that undergoes Hock rearrangement of a C–C to C–O of genotoxicity probably depends on the different metabo- bond followed by hydrolysis and Hock cleavage; (iii) the lipid lizing capacities and thus the action of different metabolites peroxyl radical of the hydroperoxides permits their facile of 4-HNE [211]. 4-HNE can be enzymatically metabolized cyclization to dioxetane and ending with dioxetane cleavage; at physiological level and cells can survive; 4-HNE can play (iv) free radical attack to 𝜔-6 PUFA on bis-allyl site yielding an important role as signaling molecule stimulating gene a free radical intermediate, that further reacts with molecular expression (mainly Nrf2) with protective functions that can oxygen to generate hydroperoxide derivatives such as 13- enhance cellular antioxidant capacity and exert adaptive HPODE or 15-HPETE. The abstraction of an allylic hydrogen response when 4-HNE level is low; under this circumstances Oxidative Medicine and Cellular Longevity 9

∙ ∙ Radical PUFA Radical lipoic acid + + H H

∙ ∙ 9-Lipid radical 13-Lipid radical

O2 O2

∙ ∙ 9-Peroxyl radical 13-Peroxyl radical

+ + H H Peroxycyclization 1 2 3

∙ ∙ ∙ 9-Hydroperoxyl radical 9, 10 dioxetane 13-Hydroperoxyl radical 13-Alkoxyl radical

O2 O2 O2 Cyclization ∙ 9-Alkoxyl radical Peroxy dioxetane Hydroperoxyl dioxetane 4 5 Fragmentation

𝛽-Scission Rearrangement 4-HPNE 4-HPNE Fragmentation Reduction

4-HNE 4-HNE 4-HNE 4-HNE 4-HNE

Figure 5: Nonenzymatic 4-HNE production. Initial abstraction of bisallylic hydrogen of lipoic acid (LA) produces fatty radicals. 4-HNE formation starting with 9- and 13-hydroperoxyoctadecadienoate (HPODE) (red and blue pathways, resp.). 4-HNE is generated by beta- scission of a hydroxyalkoxy radical that is produced after cyclization of alkoxy radical in the presence of transition metal ions and two molecules of oxygen; this reaction involves hydrogen abstraction (1). Peroxy radical cyclizes to form a dioxetane which is oxygenated to peroxy-dioxetane that is fragmented and after two hydrogen abstractions produce 4-HNE (2). Hydroperoxyl radical is oxygenated to dioxetane that is further fragmented to produce 4-hydroperoxy-2E-nonenal (4-HPNE), an immediate precursor of 4-HNE (3). Bicyclic 2+ endoperoxides react with reduced form of transition metal, such as iron (Fe ) to produce alkoxyl radicals which after reaction with oxygen + (O2), hydrogen abstraction (H ), and fragmentation produce 4-HNE (4). Alkoxyl radical after cyclization, oxygenation, hydrogen abstraction, oxidation of transition metal, hydrolysis, and rearrangement yields 4-HNE (5). With arachidonic acid, 11- and 15- hydroperoxyeicosatetraenoic acids (HPETE) are the precursors to form 4-HNE via the analogous mechanisms.

cells can survive; 4-HNE can promote organelle and protein conjugate products can be summarized according to stress damage leading to induction of autophagy, senescence, or cell levels: (i) under physiological or low stress levels the major cycle arrest at 4-HNE medium level and cells can subsist; and 4-HNE detoxification step is conjugation with GSH to yield finally 4-HNE induces apoptosis or necrosis programmed glutathionyl-HNE (GS-HNE) or glutathionyl-lactone (GS- cell death at 4-HNE high or very high level, respectively, )lactone (cyclic ester 4-HNE- form) followed by NADH- and cells die. These processes eventually lead to molecular dependent alcohol dehydrogenase (ADH-)catalysed reduc- cell damage which may facilitate development of various tion to glutathionyl-DNH (GS-DNH) and/or aldehyde dehy- pathological states. High levels of 4-HNE can also react with drogenase (ALDH-)catalysed oxidation to glutathionyl-HNA proteins and/or DNA to form adducts resulting in a variety (GS-HNA); (ii) at moderate stress levels, 4-HNE undergoes of cytotoxic and genotoxic consequences (Figure 6). aldehyde dehydrogenase (ALDH-)catalysed oxidation yield- ing HNA, that may be further metabolized in mitochondria 4-HNE Metabolism.Themaingoaloftherapidintracellular through beta-oxidation by cytochrome P450 to form 9- metabolism of 4-HNE in mammalian cells is to protect hydroxy-HNA; and (iii) at high stress levels, 4-HNE is proteins from modification by aldehydic lipid peroxida- metabolized by ADH (that belongs to the aldo-keto reductase tion products [212]. The biochemical routes of 4-HNE (AKR) superfamily) to produce DNH [131, 196, 202, 212, 213] metabolism that lead to the formation of corresponding (Figure 4). By disrupting the Gsta4 gene that encodes the alcohol 1,4-dihydroxy-2-nonene (DHN), corresponding acid alpha class glutathione s-transferase (GST) isozyme GSTA4- 4-hydroxy-2-nonenoic acid (HNA), and HNE-glutathione 4inmiceshowedthatGSTA4-4playsamajorrolein 10 Oxidative Medicine and Cellular Longevity

4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE 4-HNE Physiological Low Medium High Very high levels levels levels levels levels

Cell signaling Cell signaling Adducts and Irreversible cell and response protein damage apoptosis injury/damage 4-HNE is to stress metabolized Autophagy, Development of Programmed Cellular senescence, or pathological necrosis cell antioxidant cell cycle arrest states death induction

Cell survive Cell survive Cell subsist Cell die Cell die

Figure 6: 4-HNE promotes cell survival or induces cell death. Depending on cell type, damage/repair capacities and cellular metabolic circumstances 4-HNE can promote cell survival or induce cell death. 4-HNE at physiological levels is enzymatically metabolized and at low levels plays an important role as signaling molecule stimulating gene expression, enhance cellular antioxidant capacity and exert adaptive response; at medium levels organelle and protein damage lead to induction of autophagy, senescence, or cell cycle arrest and at high or very high levels promote adducts formation and apoptosis or necrosis cell death, respectively.

protectingcellsfromthetoxiceffectsofoxidantchemicalsby with two redox-active cysteine residues (-Cys-Gly-Pro-Cys-) attenuating the accumulation of 4-HNE [214]. Overexpres- in its active center; oxidized Trx is reduced back to the sion and inhibition of ALDH activity reduce and increase, active form of Trx by Trx reductase (TrxR) in the presence respectively, the 4-HNE toxicity and 4-HNE-protein adducts of NADPH [231]; 4-HNE can upregulate Trx/TrxR [220, levels in cell culture [215, 216]. 221, 232]; (iii) glutamate cystein ligase (GCL) is a major determinant enzyme in GSH synthesis [233, 234]. 4-HNE can upregulate GCL [235–239]. 2.5.1. 4-HNE as Signaling Molecule. At moderate concentra- Involvement of AP-1 transcription factor in 4-HNE- tion, when the basal level of antioxidant enzymes cannot induced cell signaling has been demonstrated by several be sufficient to neutralize 4-HNE, cells can survive dueto studies which showed an AP-1 upregulation by 4-HNE [240– 4-HNE may regulate several transcription factors sensible 243]. Activation of AP-1 binding may lead to the 4-HNE- to stress such as nuclear factor erythroid 2-related factor 2 inducedincreaseinGSHcontent[239]. AP-1 is a dimer con- (Nrf2), activating protein-1 (AP-1), NF-𝜅B, and peroxisome- sisting of basic region-leucine zipper proteins from the Jun proliferator-activated receptors (PPAR). It also activates and Fos subfamilies. AP-1 transcription factors control cell stress response pathways such as mitogen-activated protein proliferation, survival, and death. Growth factors, cytokines, kinases (MAPK), EGFR/Akt pathways, and protein kinase cellular stress, and many other stimuli activate AP-1 [244, C. Different labs demonstrated the 4-HNE-dependent induc- 245]. tion of Nrf2, a primary sensor and oxidative stress regulator NF-𝜅B is a dimeric transcription factor that regulates [217–221]. Also administration of the Nrf2-ARE activators diverse biological processes, including immune responses, protect from 4-HNE toxicity [222]. Under physiological con- inflammation, cell proliferation, and apoptosis. The NF- ditions, Nrf2 is sequestered in the cytoplasm by the repressor 𝜅Bproteincomplexisretainedinaninactivestateinthe protein Keap1, but in response to oxidant stimuli Nrf2 is cytoplasm by binding to inhibitory proteins I𝜅Bs family activatedandtranslocatedintothenucleuswheremediatethe [246]. Various cellular stimuli, such as oxidative stress, I𝜅Bs transcription of antioxidant/cytoprotective genes by binding are phosphorylated, making them susceptible to degradation to the antioxidant-response element (ARE) within DNA by the ubiquitin-proteasome system. This results in nuclear [223]. The Nrf2-ARE pathway has essential role in different translocation of NF-𝜅B complex where it can bind to various pathological states such as neurodegenerative diseases [223], promoter areas of its target genes and induce gene tran- cancer [224], diabetes [225], and infectious disease [226]. The scription of the corresponding genes [246, 247], most of main genes regulated by 4-HNE- induced Nrf2-ARE pathway which are implicated in the regulation of inflammation. 4- are as follows: (i) HO-1, an antioxidant protein that catalyzes HNE can activate or inhibit NF-𝜅B depending on the type the degradation of heme to biliverdin, which is then degraded of cells used. For example, 4-HNE inhibited the activity of to bilirubin; both biliverdin and bilirubin have antioxidant NF-𝜅B in hepatocytes [165], cortical neurons [248], ARPE- properties [227]; 4-HNE can upregulate HO-1 [217, 220, 221, 19 human retinal pigment epithelial cells [249], Kupffer 228–230]; (ii) thioredoxin (Trx) and thioredoxin reductase cells [250], human aortic endothelial cells [251], human (TrxR); Trx is a small (13 kDa) antioxidant ubiquitous protein colorectal carcinoma, and lung carcinoma cell [252]. On the Oxidative Medicine and Cellular Longevity 11 contrary, 4-HNE induced activity of NF-𝜅Binmacrophages the protective effects of MAPK activation via GSH induction [253], vascular smooth muscle cells [254], PC12 cells [255], because the activation of the ERK pathway is involved in GCL optic nerve head astrocytes [256], human osteoarthritic (the rate-limiting enzyme in de novo glutathione (GSH) chondrocytes [257], human fibroblasts [258], and human synthesis) regulation in rat cells [273] while the JNK pathways monocytic lineage cells [259]. appear to be involved in human HBE-1 cells [274]. PPARs comprise three subtypes (PPAR𝛼, 𝛽/𝛿,and𝛾) In human monocytes, 4-HNE was shown to significantly to form a nuclear receptor superfamily. PPARs act as key inhibit p38 and ERK activity, which resulted in inhibition of transcriptional regulators of lipid metabolism, mitochondrial TNF and interleukin-1beta production in response to LPS. biogenesis, and antioxidant defense [260, 261]. PPARs inter- The data suggest that 4-HNE, at nontoxic concentrations, has action/modulation with 4-HNE has been reviewed [262]. anti-inflammatory properties [275]. In human osteoarthritic 4-HNE increased PPAR-𝛾 gene expression and accelerated osteoblasts, 4-HNE also showed a significant (approximately adiponectin protein degradation in adipocytes [263]; expres- 70%) decrease of TNF-𝛼-induced IL-6 mRNA expression via sion of PPAR-𝛾 was induced in HL-60 and U937 cells by the NF-𝜅B signaling pathway. However, only p38 MAPK and 4-HNE treatment [264], whereas in the colon cancer cell JNK1/2 were activated, but not ERK1/2 [276], while 4-HNE (CaCo-2) PPAR𝛾 protein expression was not induced after 4- also induced COX-2 expression and prostaglandin E2 (PGE2) HNE treatment [265]; 4-HNE increased PPAR𝛾2 expression release [257, 276]. in C2C12 cells [266]. PPAR-𝛽/𝛿 is activated by 4-HNE in 3T3- On the other hand, 4-HNE mediated depletion of intra- L1 preadipocytes cells [267]. 4-HNE activates PPAR-𝛿 and cellular thiols, protein tyrosine phosphorylation, MAPK amplifies insulin secretion in INS-1E 𝛽-cells [152]. (JNK, ERK, and p38) activation, and modulates integrin MAP kinases familycanbeactivatedinresponseto resulting in reorganization of cytoskeletal, focal adhesion diverse stimuli such as oxidative stress, lipopolysaccharides, proteins, and barrier dysfunction in lung microvascular inflammatory cytokines, growth factors, or endoplasmic endothelial cells [277]. Results suggest that activation and reticulum (ER) stress and are involved in several cellu- phosphorylation of MAP kinases (JNK, ERK, and p38) play lar responses like cell proliferation and/or differentiation, an important role in 4-HNE mediated toxicity and cell inflammation, proteasomal-mediated protein degradation, death in mouse embryonic fibroblasts (MEF), and absence and apoptosis. Members of the major mitogen-activated of GSTA4–4 potentiates the cytotoxic effects of 4-HNE. The protein kinase (MAPK) subfamilies are the extracellular increase of apoptosis in Gsta4 null MEF by 4-HNE was asso- signal-regulated kinase (ERK), p38, and Jun N-terminal ciated with the enhanced accumulation of 4-HNE-protein kinase (JNK) subfamilies. The mechanism by which MAPK adducts, DNA damage, and the activation of caspases-3, signaling cascades are activated by 4-HNE is not well known. -8, and -9 [214]. 4-HNE upregulates and phosphorylates For example, activation of different MAPK under various cytosolic phospholipase A-2 (cPLA-2) in cultured microglial stimuli can affect both apoptotic and prosurvival signaling. cell line (Ra2) via the ERK and p38 MAPK pathways [278]. In corneal epithelial cells, 4-HNE caused a time-dependent cPLA is a proinflammatory enzyme that stimulate AA- release induction of HO-1 mRNA and protein via modification by hydrolyzes glycerophospholipids with AA in the sn-2 and activation of Erk1/2, JNK and p38 MAP kinases, as position. well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of Matrix metalloproteinases (MMPs) constitute a large p38 blocked 4-HNE-induced HO-1 expression; inhibition of group of endoproteases that are not only able to cleave all Erk1/2 and, to a lesser extent, JNK and PI3 K/Akt suppressed protein components of the extracellular matrix but also to 4-HNE-induced HO-1 [268]. 4-HNE also stimulated Erk1/2, activate or inactivate many other signaling molecules, such JNK, p38, and PI3 kinase in keratinocyte, and the inhibitors as receptors, adhesion molecules, and growth factors [279]. of these enzymes suppressed 4-HNE-induced expression of 4-HNE induced MMP-9 production in macrophages [280] HO-1 [269]. In PC12 cells, 4-HNE treatment induced ERK, and MMP-2 in vascular smooth muscle cells (VSMC) [281] JNK, and p38 MAPK activation as well as induced the via activation of ERK and p38 MAPK pathways, consequently expression of HO-1. Addition of p38 MAPK specific inhibitor leading to plaque instability in atherosclerosis. 4-HNE also SB203580 attenuated HO-1 upregulation; these results indi- enhances MMP-2 production in VSMC via mitochondrial cate that 4-HNE-induced transient p38 MAPK activation ROS-mediated activation of the Akt/NF-kappaB signaling may serve as an upstream negative regulator of ER stress pathways [254]. In osteoarthritic (OA) synovial cells, 4-HNE and confer adaptive cytoprotection against 4-HNE-mediated induced MMP-13 mainly through activation of p38 MAPK cell injury [228]. In rat liver epithelial RL34 cells, 4-HNE [282]. upregulates the cyclooxygenase-2 (COX-2, which plays a key Akt(a.k.aproteinkinaseBorPKB)comprises three closely role in conversion of free arachidonic acid to PGs) expression related isoforms Akt1, Akt2, and Akt3 (or PKB𝛼/𝛽/𝛾 resp.), by the stabilization of COX-2 mRNA via activation of the which play a role in the regulation of cell proliferation, p38 MAPK pathway [270]. In human hepatic stellate cells survival, and metabolism. Dysregulation of Akt leads to (hHSC), 4-HNE forms adducts with JNK and this event diseases such as cancer, diabetes, and cardiovascular and leads to JNK nuclear translocation and activation as well neurological diseases [283]. Under conditions of enhanced as to c-jun and AP-1 induction [271]. In human bronchial oxidative stress, a major cellular response is the activa- epithelial cells, 4-HNE downmodulates the protein-tyrosine tion of the Akt pathway that involves the oxidation and phosphatase SH2 domain containing phosphatase-1 (SHP-1) subsequent inactivation of PTEN (phosphatase and tensin which negatively regulates JNK activity [272]. We can also see homolog deleted on chromosome 10), a tumor suppressor 12 Oxidative Medicine and Cellular Longevity andprimaryregulatorofAkt[284]. Recent studies have early upregulation of monocyte chemoattractant protein 1 also demonstrated that activation of PI3 K/Akt signaling by (MCP-1) release occurs in response to low 4-HNE concen- 4-HNE occurs via modification and inhibition of PTEN, trations, most likely through of the increase in the activity a regulatory protein that suppresses Akt2 activity, which of PKC-𝛽Iand𝛽II classic isoforms, while the activation of is selectively phosphorylated by 4-HNE in both cellular PKC-𝛿 appeared to be involved in LPS-stimulated cells [299]. human hepatocellular carcinoma cell line (HepG2) [285] Treatment of macrophages with 4-HNE, cell-permeable and animal models (ethanol-fed mice) [286]. In HepG2 esters of glutathionyl-4-hydroxynonenal (GS-HNE) and cells, 4-HNE inhibits H2O2-mediated activation of the Akt glutathionyl-1,4-dihydroxynonane (GS-DHN) activated NF- pathway in leading to phosphorylation of Akt1 but not Akt2, 𝜅B and PLC/PKC. Aldolase reductase catalyzes the reduction decreased cell proliferation, and decreased expression of of GS-HNE to GS-DHN. AR inhibition/ablation prevented cyclin D1 [287]. In retinal pigment epithelial (RPE) cells, at PLC, PKC, and IKKalpha/beta, and NF-𝜅B activation caused lower concentrations 4-HNE triggered phosphorylation of by 4-HNE and GS-HNE, but not by GS-DHN, suggests a epidermal growth factor receptor (EGFR) and activation of its novel role for a reduced glutathione-lipid aldehyde conjugate downstream signaling components ERK1/2 and Akt; this led (such as GS-DHN) as an obligatory mediator of ROS-induced to protective mechanism against oxidative stress [288]. Akt- cytotoxicity [300]. induced activity by 4-HNE promotes cell survival through induction of HO-1 mRNA and protein in corneal epithelial 2.5.2. Effect of 4-HNE on Autophagy. One of the most impor- cells [268],andinkeratinocyte[269]. The inhibitors of Akt tant processes for maintaining normal metabolic and redox suppressed 4-HNE-induced expression of HO-1. signaling, through degradation of damaged proteins and Protein kinases C (PKCs) are a family of multifunctional organelles, is autophagy-lysosomal pathway [301]. 4-HNE enzymes that play crucial roles in the transduction of many can promote protein-adducts leading to protein damage and cellular signals such as control of cell proliferation, survival, to induction of autophagy-lysosomal pathway [302], a pro- and transformation by phosphorylating various targets. The cess that is increased by treatment with an autophagy stimula- PKC family consists of three different groups: conventional tor, rapamycin. If autophagy is blocked with a PI3 K inhibitor, (𝛼, 𝛽1, 𝛽2, and 𝛾), novel (𝛿, 𝜀, 𝜂,and𝜃), and atypical (𝜁 3-methyladenine, apoptotic cell death occurs [301, 302]. Sev- and 𝜆/𝜏). Conventional and novel PKC isoforms are lipid- eral mechanisms by which 4-HNE induces autophagy have sensitive enzymes and calcium-dependent and are usually been reported. For example, 4-HNE promotes the formation activated by growth factors through stimulation of phos- of protein adducts that accumulate in the endoplasmic pholipase C (PLC) which hydrolyzes phosphatidylinositol- reticulum (ER) and led to autophagy in rat aortic smooth 4,5-bisphosphate (PIP2) to generate inositol triphosphate muscle cells, through selective activation of the PKR-like ER (IP3) and DAG [6, 289]. Cells can express more than one kinase (PERK) pathway accompanied by JNK activation, the PKC isoform, and individual PKCs can mediate different upregulation of the HO-1, increased microtubule-associated biological processes. For example, in human promyelo- protein 1 light chain 3 (LC3) formation, and maintenance of cytic leukemia (HL-60) cells [290–292]andratneutrophils cell viability under conditions of excessive 4-HNE-protein [293] 4-HNE induced a significant increase of PLC activity, adducts accumulation [303]. In differentiated SH-SY5Y neu- which should result in an increased production of IP3 and roblastoma cells, glucose-dependent autophagy serves as a DAG, known to stimulate PKC [289]. Phagocytes, such protective mechanism in response to 4-HNE because low as granulocytes and monocytes/macrophages which engulf 4-HNE-concentrations increased autophagy and induced microbial intruders and effectively kill and eradicate the concentration dependent CASP3/caspase-3 activation and foreign bodies, contain a membrane-associated NADPH cell death. Additionally inhibition of glucose metabolism by oxidase that produces superoxide leading to other ROS with 2-deoxyglucose and glycolysis by koningic acid, a GAPDH, microbicidal, tumoricidal, and inflammatory activities [294]. inhibitor, led to autophagy inhibition and increased CASP3 In RAW 264.7 mouse macrophage cells, 4-HNE exhibited activation and cell death [304]. On the contrary, phagocy- a concentration-dependent inhibition of ROS by adduction tosis of 4-HNE- and MDA-modified photoreceptor outer to PKC, a protein vital in the assembly and activation of segments (POS) induced a marked reduction of autophagic NADPH oxidase [295]. In rat hepatocyte PKC- isoforms activity by 40% in retinal pigment epithelium (RPE) cells, activity is differentially regulated by concentrations 4-HNE. which may contribute to RPE cell dysfunction and degener- For example, PKC-𝛼 activity was decreased in a dose- ation. In contrast, unmodified POS had no significant effect dependent manner by all concentrations of 4-HNE, while low on autophagy [305]. concentrations of 4-HNE increased PKC 𝛽Iand,toamuch greater extent, PKC 𝛽II activities. By contrast, they were unaf- fected or even inhibited by higher concentrations of 4-HNE. 2.5.3. Effect of 4-HNE on Senescence. Cellular senescence, This PKC-dependent- 4-HNE regulation could be involved defined as arrest during the cell cycle (G0), is involved in the traffic of secretory glycoproteins [296]. In NT2 neu- in the complex process of the biological aging of tissues, rons, low 4-HNE concentrations (similar to concentrations organs, and organisms. Senescence is driven by many factors detected in AD brain tissue) induced a 2–6 fold increase including oxidative stress, the DNA damage/repair response, of intracellular amyloid 𝛽-protein (A𝛽)productionthat inflammation, mitogenic signals, and telomere shortening. was concomitant with selective activation of 𝛽Iand𝛽II Telomeres are considered a “biological clock” of the cell PKC isoforms [297, 298]. In macrophages, a marked and andareshortenedbyeachcelldivisionuntilacritical Oxidative Medicine and Cellular Longevity 13 length is reached and dysfunction ensues. Rapid telomere to transition of S to G2 and cyclin B leads G2 to M phases shortening may indicate a very high cellular activity. DNA- [321, 322]. The promitotic factor Cdc25 stimulates cell cycle repair pathways are then recruited and cells enter senescence, progression through the activation of cyclin A-Cdk1, cyclin losing their capacity to proliferate. In addition to cell division, B-Cdk1, and cyclin E-Cdk2 for entry into M phase by remov- factors causing telomere shortening include DNA damage, ing the inhibitory phosphorylation on Cdk1 and Cdk2. On inflammation, and oxidative stress306 [ ]. Activation of a the contrary, the anti-mitotic factor (p21, p27,p57) inhibit cell DNA damage response including formation of DNA damage cycle progression through inhibition of cyclin A–Cdk1, cyclin foci containing activated H2A.X (𝛾-histone 2A.X) at either B–Cdk1, cyclin E–Cdk2 and cyclin D–Cdk4/6 [321–323]. In uncapped telomeres or persistent DNA strand breaks is the response to 4-HNE, the expression of key components of cell major trigger of cell senescence. 𝛾H2AX is a sensitive marker cycle can be modulated and cells are arrested at G1 or G2. of DNA damage, particularly induction of DNA double- Several studies showed that in general 4-HNE may induce strand breaks [307]. The length of telomeres depends on the cell cycle arrest in malignant cell and inhibition or decrease telomerase activity and the catalytic subunit of telomerase of cell proliferation. For example, treatment of HL-60 cells (hTERT) which is strongly upregulated in most human can- with 4-HNE (1 𝜇M) causes a p53-independent increase of cers [308], and the major consequence of the reactivation of p21 expression, RB dephosphorylation, progressive reduction telomerase activity is that tumor cells escape from senescence. in the amount of free E2F bound to DNA, and a relative The expression of c-myc (an activator),mad-1(a repressor) increase in E2F complexes at higher molecular weights with and sp-1 (anactivator/repressor),whichhavebeenshown repressive activity decrease of E2F complexes [324], and to activate hTERT transcription. The formation of 4-HNE- decrease of cyclin D1, cyclin D2, and cyclin A [325]. In human proteins adducts in general increased as a function of age erythroleukemia cells (K562), 4-HNE treatment increased [309]. Quantitative evaluation showed that the majority of p53 and p21 expression and decreased expression of cyclin senescent hepatocytes (as measured by 𝛾-H2A.X) were also D2. The additional decrease of A- and B-cyclin suggests that positive for 4-HNE [310, 311]. 4-HNE can induce premature the S- and G2-phase were also retarded contributing to the senescence by a direct suppression of telomerase activity overall slowdown of the cycle [326]. In human breast cancer affecting the expression of hTERT. In endothelial cells (EC) cells (MCF7) the increase in endogenous levels of 4-HNE isolated and cultured from arterial segments of patients with caused by treatment with conjugated linoleic acid (CLA) severe coronary artery disease, chronic treatment with an resulted in the inhibition of cell proliferation through a p53- antioxidant (that significantly decreased the levels of lipid dependent mechanism [327]. In human osteosarcoma cells peroxidation, that is, 4-HNE expression) N-acetyl-cystein, (HOS), 4-HNE treatment declined gradually the proportion NAC, significantly delayed cellular senescence via decrease of of cells in mitosis, inhibited proliferation and differentiation, DNA damage marker (𝛾H2AX), decrease of nuclear p53, and and increased apoptosis [328]. In malignant cells like hep- increase in hTERT activity [312]. In three human leukemic atome cells, with a below-normal content of PUFAs and very cell lines (HL-60, U937, and ML-1) [313] and in colon high expression of aldehyde dehydrogenase-3 (ADH3) which cancer cells (Caco-2 and HT-29) [314], telomerase activity metabolize 4-HNE to DNH, the inhibitory effects of 4-HNE and hTERT expression were downregulated by 4-HNE, as a on cell proliferation are lower, but the inhibition of ADH3 consequenceofdownregulationofc-myc mRNA expression resulted in an increase in the quantity of aldehyde in the cells and c-Myc DNA binding activity as well as upregulation of and inhibit cell proliferation through the MAPK pathway by mad-1 mRNA expression and Mad-1 DNA binding activity. reduction of pRaf-1 and pERK1,2 [329, 330]. Moreover, 4- On the other hand, 4-HNE may induce cellular senescence HNE has also antiproliferative/differentiative effect mainly in through activation of critical cell cycle sentinels that mediate malignant cell, by affecting the expression of key genes, such this process, such as the tumor suppressor proteins p53 as oncogenes (e.g., c-myc and c-myb)andcyclins.Inthree (see below),whichiswellknowntoplayacentralrolein human leukemic cell lines (HL-60, U937,and ML-1) [313]and senescence [315–320]. p53 protects cells of oxidative stress in colon cancer cells [265, 314], cell proliferation was inhibited and promotes DNA repair. However, when in the cells the by 4-HNE, as a consequence of downregulation of c-myc extent of damage overwhelms repair capacities, p53 induces mRNA. 4-HNE mediated inhibition of cell proliferation in cell death [315–319]. All these data thus confirmed a cell- the HL-60 cell line by downregulation of Notch1, which is specific association between senescence and 4-HNE. involved in expression of cyclin D1 and c-Myc [331]. In SK- N-BE human neuroblastoma cells, 4-HNE upregulated p53 family gene expression and p53 gene targets p21 and bax, and 2.5.4. Effect of 4-HNE on Cell Cycle and Proliferation. In cell theconsequentreductioninS-phasecellsandtheincreased cycle the transition of different phases is driven by several apoptotic cell proportion; 4-HNE also reduced cyclin D2 phase-specific cyclin-CDK (cyclin-dependent kinase) com- expression [332]. In HepG2 cells, 4-HNE decreased both cell plexes which previously have been activated. In response to survival and proliferation as evidenced by MTT assays and mitogens, cyclin D is activated and phosphorylate retinoblas- EdU incorporation as well as decreased expression of cyclin toma protein (RB) which leads to activation of E2F proteins D1 and 𝛽-catenin [287]. In K562 cells [333], HL-60 human and the expression of E2F-responsive genes inducing cells to leukemic cell line [334], and murine erythroleukemia (MEL) reenterthecellcyclefromquiescencecalledG0,toG1.Activa- cells [335], 4-HNE inhibited c-myc expression; a oncogene tion of E2F leads to the transcription of cyclin E for transition is involved in the regulation of cellular multiplication and from G1 to S phase. Subsequent expression of cyclin A leads transformation (see review of Barrera and co-workers [336]). 14 Oxidative Medicine and Cellular Longevity

All these effects increased the proportion of G0/G1 cells, The alternative to apoptosis or programmed cell death is indicating cell cycle arrest at G1 [324, 325, 336, 337]. 4- necrosis or nonprogrammed cell death, which is considered HNE-induced G2/M cell cycle arrest was via p21 through a to be a toxic process where the cell is a passive victim and mechanism (s) that is independent of p53. The cell cycle arrest follows an energy-independent mode of death. Depending leads to apoptotic cell death [338]. Enterococcus faecalis— on the cell type, DNA damage/repair capacity or cellular infected macrophages produce 4-HNE. This electrophile, metabolic circumstances 4-HNE can activate proliferative when purified, mediated bystander effects in colonic epithe- signaling for cell division and promote cell survival or “stop” lial cells by generating 𝛾H2AX foci and inducing G2/M cell cell division, and after prolonged arrest, cells die from apopto- cycle arrest. 4-HNE was also associated with mitotic spindle sis. 4-HNE may induce these processes by modulating several damage, activation of stathmin, cytokinesis failure, and the transcription factors sensible to stress such as Nrf2, AP-1, NF- development of tetraploid [339]. In PC3 prostate cancer cell, 𝜅B, and PPAR or by modulating several signaling pathways, 4-HNE induced G2/M cell cycle arrest by decreasing p-Cdc2 including MAPK (p38, Erk, and JNK), protein kinase B, (entry into M phase is determined by activation of the Cdc2 protein kinase C isoforms, cell-cycle regulators, receptor protein kinase, which requires Cdc2 dephosphorylation); tyrosine kinases, and caspases. Depending on 4-HNE con- increased amount of p-H2A.X indicated that 4-HNE induced centrations the cells “end” their lives by apoptosis or necrosis. apoptotic cell death after a G2/M accumulation [340]. For example, the cytotoxicity of 4-HNE to HepG2 cells was In an opposite way, different studies indicated that 4- evaluated by MTT assay. 4-HNE concentrations ranging from HNE can promote cell proliferation in normal cells, mainly 10 to 100 𝜇M gradually decreased cell viability corresponding by upregulation of cyclin or E2F. In cultured primary cortical to an IC50 value of 53 ± 2.39 𝜇M. 4-HNE concentrations neurons, 4-HNE increased the protein levels of phospho- of 5–40 𝜇M caused apoptotic cell death (measured by flow p53 and cell cycle-related proteins (cyclin D3, cyclin D1, cytometry, caspase-3 activation, and PARP cleavage). Finally, and CDC25A), caspase-3 activation, PARP cleavage, calpain a significant increase in necrotic cell population, that is, 31.8% activation, serine/threonine kinase 3 (Stk3), and sphingosine and 55.4%, was observed in cells treated with 80 and 100 𝜇M phosphate lyase 1 (Sgpl1) upregulation. NAC decreased cell of 4-HNE, respectively [350]. These results show that 4-HNE death [341]. In smooth muscle cells (SMCs), treatment with induces apoptosis at low concentration and necrosis at high 4-HNE enhanced cyclin D1 expression and activation of the concentration. ERK signaling pathway, which were stronger in young SMCs The two main pathways of apoptosis are extrinsic and compared with aged SMCs [342]. 4-HNE induced vascular intrinsic pathways. The extrinsic signaling pathways that smooth muscle cell proliferation [142, 343]. Aldose reductase initiate apoptosis involve transmembrane receptor-mediated (AR) efficiently reduces 4-HNE and GS-HNE. Inhibition interactions. This pathway is triggered by the binding of death of AR can arrest cell cycle at S phase. In VSMC cells, the ligands of the tumor necrosis factor (TNF) family to their inhibition of AR prevents high glucose (HG-) and/or TNF- appropriate death receptors (DRs) on the cell surface; best- alpha-induced VSMC proliferation by accumulating cells at characterized ligands and corresponding death receptors the G1 phase of the cell cycle. Treatment of VSMC with 4- includeFasL/FasRandTNF-𝛼/TNFR1 [351, 352]. The intrin- HNE or its glutathione conjugate (glutathionyl (GS-)HNE) or sic signaling pathways that initiate apoptosis involve a diverse AR-catalyzed product of GS-HNE, GS-1,4-dihydroxynonane array of non-receptor-mediated stimuli. The proapoptotic resulted in increased E2F-1 expression. Inhibition of AR member of the Bcl-2 family of proteins, such as Bax, per- prevented 4-HNE- or GS-HNE-induced upregulation of E2F- meabilizes the outer mitochondrial membrane. This allows 1. Collectively, these results show that AR could regulate redistribution of cytochrome c from the mitochondrial inter- HG- and TNF-alpha-induced VSMC proliferation by altering membrane space into the cytoplasm, where it causes activa- the activation of G1/S-phase proteins such as E2F-1, cdks, tion of caspase proteases and, subsequently, cell death [352, and cyclins [344]. In airway smooth muscle cells, 4-HNE 353]. Each apoptosis pathway requires specific triggering is mitogenic by increasing cyclin D1 activity through ERK signals to begin an energy-dependent cascade of molecular signaling pathway [345]. events. Each pathway activates its own initiator caspase (8, The differential effect of 4-HNE on cell proliferation in 9)whichinturnwillactivatetheexecutionercaspase-3 both malignant and nonmalignant cells may be the conse- [352]. The execution pathway results in characteristic cyto- quence of lower aldehyde-metabolizing enzymes, deregula- morphological features including cell shrinkage, chromatin tion of antioxidant defenses, and mitochondrial metabolism condensation, formation of cytoplasmic blebs and apoptotic alteration [132, 346], so that malignant cells are more vulner- bodies, and finally phagocytosis of the apoptotic bodies by able to further oxidative stress induced by exogenous ROS- adjacent parenchymal cells, neoplastic cells or macrophages generating agents or inhibitors of the antioxidant systems [352, 353]. A multitude of mechanisms are employed by [347–349]. p53 to ensure efficient induction of apoptosis in a stage-, tissue-, and stress-signal-specific manner [354]. 4-HNE- mediated activation of p53 may be one of the mechanisms 2.5.5. 4-HNE-Induced Apoptosis and Necrosis. Apoptosis is responsible for 4-HNE-induced apoptosis reported in many essential programmed cell death process for cells, and its cell types. For example, in SH-SY5Y cells 4-HNE-induced dysregulation results in too little cell death which may oxidative stress was associated with increased transcriptional contribute to carcinogenesis, or too much cell death which andtranslationalexpressionsofBaxandp53;theseevents may be a component in the pathogenesis of several diseases. trigger other processes, ending in cell death [355]. In RPE Oxidative Medicine and Cellular Longevity 15 cells, 4-HNE causes induction, phosphorylation, and nuclear signaling 4 (RGS4) can be modified by 4-HNE. RGS4, like accumulation of p53 which is accompanied with downregu- other RGS proteins, is responsible for temporally regulating lation of MDM2, a negative regulator of the p53 by blocking G-protein coupled receptor signaling by increasing the p53 transcriptional activity directly and mediating in the intrinsic GTPase activity of G𝛼 subunit of the heterotrimeric p53-degradation. Associated proapoptotic genes Bax, p21, signaling complex. 4-HNE modification of RGS4 at cysteine and JNK, which are all signaling components p53-mediated residues during oxidative stress can disrupt RGS4 activity pathway of apoptosis, are activated in response to exposure to and alter signaling from stressed cells. Possibly 4-HNE acts 4-HNE. The induction of p53 by 4-HNE can be inhibited by as an internal control for aberrant signaling due to excess the overexpression of either hGSTA4 (in RPE cells) or mGsta4 RGS4 activity in a variety of pathologies where oxidative (in mice) which accelerates disposition of 4-HNE [356]. stress is a strong component [362]. Our lab has reported that In CRL25714 cell, 4-HNE induced dose-dependent increase 4-HNE can affect protein synthesis rates by forming adduct in the expression of p53 in the cytoplasmic and nuclear with eEF2 (see below—cumene hydroperoxide-induced lipid compartments and increase in the expression of Bax [357]. peroxidation). Large lists of peptides and proteins known to In human osteoarthritic chondrocytes, 4-HNE treatment led be modified by 4-HNE are given in the reviews [76, 104, 363] to p53 upregulation, caspase-8, -9, and -3 activation, Bcl- and including glutathione, carnosine, enzymatic proteins, 2 downregulation, Bax upregulation, cytochrome c-induced carriers proteins, membrane transport proteins, receptor release from mitochondria, poly (ADP-ribose) polymerase proteins, cytoskeletal proteins, chaperones, mitochondrial cleavage, DNA fragmentation, Fas/CD95 upregulation, Akt upcoupling proteins, transcription and protein synthesis inhibition, and energy depletion. All these effects were factors, and antioxidant proteins. inhibited by an antioxidant, N-acetyl-cysteine [358]. It has been reported that 4-HNE also could react 4-HNE can induce apoptosis through the death receptor with deoxyguanosine to form two pairs of diastereomeres Fas (CD95-)mediated extrinsic pathway as well as through adducts (4-HNE-dG 1,2 and 3,4) that further induced the p53-dependent intrinsic pathway. For detailed infor- DNA crosslink or DNA-protein conjugates. The mecha- mation of the molecular mechanisms involved in 4-HNE- nism involves a nucleophilic Michael addition of the NH2- induced programmed cell death see review [359]. However, group of deoxyguanosine to the CC double bond of 4- thesemechanismscanbesummarizedinthefollowing:(i)4- HNE, which yields 6-(1-hydroxyhexanyl)-8-hydroxy-1,N(2)- 󸀠 HNE is diffusible and can interact with Fas (CD95/Apo1) on propano-2 -deoxyguanosine (HNE-dG), an exocyclic adduct plasma membrane and upregulate and activate its expression [49, 133, 134]. HNE-dG adducts have been detected in human to mediate the apoptotic signaling through activation of and animal tissues. They are potentially mutagenic and downstream kinases (apoptosis signal-regulating kinase 1 or carcinogenic and can be repaired by the nucleotide excision ASK1 and JNK), which leads to activation of executioner repair (NER) pathway [364, 365]. In the presence of peroxides caspase-3 and ending in apoptosis; (ii) 4-HNE interacts with a different reaction takes place, and the stable end-product cytoplasmic p53 which causes its induction, phosphoryla- found in the reaction of 4-HNE with DNA bases is etheno- tion, and nuclear translocation. In the nucleus p53 inhibits DNA adducts because 4-HNE is converted by the peroxide transcription of antiapoptotic genes (Bcl2) and promotes to the corresponding epoxynonanal, which then reacts to transcription of proapoptotic genes (Bax) or cell cycle genes the NH2-group of guanosine followed by cyclization reaction 6 󸀠 4 (p21) leading to activation of executioner caspase-3 and to form 1, N -etheno-2 -eoxyadenosine (𝜀dA), and 3, N - 󸀠 ending in apoptosis or cell cycle arrest, respectively; (iii) 4- etheno-2 -deoxycytidine (𝜀dC). These 𝜀-adducts are elimi- HNE also activates a negative feedback on Fas activation, nated by the base excision repair (BER) pathway [49, 366]. by a mechanism involving transcription repressor death Etheno-DNA adduct levels were found to be significantly domain-associated protein (Daxx), a nuclear protein which is elevated in the affected organs of subjects with chronic associated with DNA-binding transcription factors involved pancreatitis, ulcerative colitis, and Crohn’s disease, which in stress response. 4-HNE interacts with the Daxx, bound to provide promising molecular signatures for risk prediction heat shock factor-1 (HSF1), translocates Daxx from nucleus and potential targets and biomarkers for preventive measures to cytoplasm where it binds to Fas, and inhibits activation of [367, 368]. The 4-HNE-DNA adducts in tissue could serve ASK1 to limit apoptosis. as marker for the genetic damage produced by endogenous oxidation of omega-6-PUFAs.

2.5.6. 4-HNE-Biomolecules Adducts. The preference for amino acid modification by 4-HNE is Cys ≫ His > Lys 3. The Use of Mammalian Model in Lipid resulting in covalent adducts with the protein nucleophilic Peroxidation Research: Compounds side chain [104, 131, 360, 361]. The reaction between Induced Lipid Peroxidation primary amines and 4-HNE carbonyl carbon groups yields a reversible Schiff base and the addition of thiol or amino The use of mammalian model in lipid peroxidation research compounds on 4-HNE 𝛽-carbon atom’ (C of double bond) is ideal for studying the consequences of lipid peroxidation produces the corresponding Michael adduct [49]. 4-HNE- in the context of whole organism and also to analyze their protein adducts can contribute to protein crosslinking and influence on biomarkers to gain more insight into what induce a carbonyl stress. Recently it has been shown that a controls the lipid peroxidation and how lipid peroxidation- membrane associated protein called regulator of G-protein relateddiseasesoccur.Animalmodelsusedtoinvestigatethe 16 Oxidative Medicine and Cellular Longevity

𝛼-Cumyl alcohol Cumoperoxyl Cumene hydroperoxide ∙ OH OO OOH

H3C C CH3 H3C C CH3 H3C C CH3

4 1 +

∙ 3 LH L 3 5 Cumene hydroperoxide LP initiation/propagation O2 ∙ OOH O OH

H3C C CH3 H3C C CH3 H3C C CH3 − ∙ OH LH L 1 2

n (n+1) M M Cumoxyl 𝛼-Cumyl alcohol

Figure 7: Mechanisms showing how cumene hydroperoxide produces lipophilic cumoxyl and cumoperoxyl radicals. Cumene hydroperoxide in presence of transition metal ions produces cumoxyl radical (step 1), which abstracts a hydrogen (H) from a lipid (PUFA) molecule (LH) ∙ generating cumyl alcohol and lipid radical (L ) that reacts readily with oxygen promoting the initiation or propagation of lipid peroxidation. (Step2).Cumoxylradicalcanalsoreactwithothercumenehydroperoxide molecules to yield cumyl alcohol and cumoperoxyl radical (step 3). Finally, cumoperoxil radical may abstract hydrogen (H) from the closest available lipid to produce a new cumene hydroperoxide and ∙ lipid radical (L ) which then again affects lipid peroxidation cycling (step 4). Cumoperoxyl radical may also react with oxygen to yield anew cumoxyl radical thus initiating a chain reaction (step 5). genetic, physiological, or pathological consequences of lipid made extensive use of membrane-soluble CH as a model peroxidation should try to control the intrinsic and extrin- compound for lipid hydroperoxides (LOOH), which are sic influences. Genetic background, diet, environment, and formed in the process of lipid peroxidation during oxidative health status can be strictly controlled in many model organ- stress. CH-induced lipid peroxidation in animals has been isms. Compared with other model organisms, such as worms importanttostudytheeffectoflipidperoxidationonprotein (Caenorhabditis elegans) and flies (Drosophila melanogaster), synthesis through mechanisms that involve regulation of the mammalian model is highly comparable to the human eElongation Factor 2 (eEF2). It is known that eEF2 plays a in respect to organ systems, tissues, physiologic systems, and key role as a cytoplasmic component of the protein synthesis even behavioral traits. Finally, mammalian model in LP can machinery, where it is a fundamental regulatory protein of be used as a first step toward possible development of drugs the translational elongation step that catalyzes the movement or interventions to control lipid peroxidation process and of the ribosome along the mRNA. One particularity of eEF2 prevent disease progression in humans. Various mammalian is that it is quite sensitive to oxidative stress and is specifically models have been developed to study the lipid peroxidation affected by compounds that increase lipid peroxidation, process. such as cumene hydroperoxide (CH) [370–373]. We have previously reported that cytotoxic end-products of lipid peroxidation 4-HNE and MDA are able to form adducts 3.1. Cumene Hydroperoxide-Induced Lipid Peroxidation. with eEF2 in vitro [374]andin vivo [309], demonstrating, for Cumene hydroperoxide (CH) a catalyst used in chemical the first time, that this alteration of eEF2 could contribute and pharmaceutical industry [369] is a stable organic to decline of protein synthesis, secondary to LP increase. oxidizing agent with the peroxy function group, –O–O–, The formation of these peroxide-eEF2-adducts is a possible which induces lipid peroxidation. On the existence of mechanism responsible of suboptimal hormone production transition-metal, CH can be reduced to form an alkoxyl from hypothalamic-hypophysis system (HHS) during radical, which can attack adjacent fatty acid side-chains oxidative stress and aging [375]. The protection of eEF2 to produce lipid radical and cumyl alcohol. The resulting alterations by end-products of lipid peroxidation must be lipid radical reacts with oxygen to form a lipid peroxyl specifically carried out by compounds with lipoperoxyl radical. And a lipid peroxyl radical reacts with other fatty radical-scavenging features such as melatonin. We have acid side-chains to produce a new lipid radical and lipid reported the ability of melatonin to protect against the hydroperoxide and this chain reaction continues. These lipid changes that occur in the eEF2 under conditions of lipid hydroperoxides may undergo transition-metal mediated peroxidation induced by CH, as well as decline of protein one-electron reduction and oxygenation to give lipid peroxyl synthesis rate caused by lipid peroxidation, demonstrating radicals, which trigger exacerbating rounds of free radical- that melatonin can prevent the decrease of several hormones mediated lipid peroxidation (Figure 7). In our lab we have after exposure to LP376 [ ]. In vitro studies carried out in Oxidative Medicine and Cellular Longevity 17 our lab also indicated that the antioxidants have different by several diseases has been widely utilized, indirectly impli- capacities to prevent eEF2 loss caused by CH [377, 378]. In rat cating MDA and 4-HNE in the pathogenesis of these diseases. hippocampal neurons and in response to lipid peroxidation Table 1 shows a brief extract of studies presented in the induced by exposure to CH, eEF2 subcellular localization, literature in which MDA and 4-HNE have been found to abundance, and interaction with p53 were modified379 [ ]. be significantly modified in pathological contexts. The “big” Finally, using CH-induced lipid peroxidation, we found challenge in the field of pathological processes is that it is that a unique eEF2 posttranslational modified derivative of often difficult to determine whether these lipid peroxidation- histidine (H715) known as diphthamide plays a role in the derived aldehydes are actually involved in causing the disease protection of cells against the degradation of eEF2, and it or are a consequence to it. is important to control the translation of IRES-dependent proteins XIAP and FGF2, two proteins that promote cell 5. Conclusions survival under conditions of oxidative stress [380]. Other labs have used cumene hydroperoxide as a model compound As conclusion, in this review we summarized the physio- for lipid hydroperoxides in vivo [381–385]. logical and pathophysiological role of lipid peroxides. When oxidant compounds target lipids, they can initiate the lipid 3.2. Tert Butyl Hydroperoxide. It is an organic oxidizing peroxidation process, a chain reaction that produces multiple agent containing a tertiary butyl group, commonly used in breakdown molecules, such as MDA and 4-HNE. Among industry as prooxidizing, a bleaching agent, and an initiator several substrates, proteins and DNA are particularly suscep- of polymerization. Tert butyl hydroperoxide is a strong tible to modification caused by these aldehydes. MDA and 4- free radical source and has been utilized to induce lipid HNE adducts play a critical role in multiple cellular processes peroxidation in vivo mammalian model [386–392]. and can participate in secondary deleterious reactions (e.g., crosslinking) by promoting intramolecular or intermolecular protein/DNA crosslinking that may induce profound alter- 3.3. Carbon Tetrachloride (CCl4). It is a toxic, carcinogenic ation in the biochemical properties of biomolecules, which organic compound which is used as a general solvent in may facilitate development of various pathological states. industrial degreasing operations. It is also used as pesticides Identification of specific aldehyde-modified molecules has and a chemical intermediate in the production of refrigerants. led to the determination of which selective cellular function Carbon tetrachloride has been utilized to induce lipid perox- is altered. For instance, results obtained in our lab suggest idation in vivo mammalian model [90, 393–398]. that lipid peroxidation affects protein synthesis in all tissues during aging through a mechanism involving the adduct 3.4. Quinolinic Acid (QA). It is a neuroactive metabolite formation of MDA and 4-HNE with elongation factor-2. ofthekynureninepathway.Itisnormallypresentedin However, these molecules seem to have a dual behavior, nanomolar concentrations in human brain and cerebrospinal since cell response can tend to enhance survival or promote fluid (CSF) and is often implicated in the pathogenesis of cell death, depending of their cellular level and the pathway avarietyofhumanneurologicaldiseases[399]. QA has activated by them. been used to induce lipid peroxidation mediated by hydroxyl radicals in vivo mammalian models [400–405]. Conflict of Interests

3.5. Transition Metals Ions. Theyareessentialelements The authors declare no competing financial interests. which, under certain conditions, can have prooxidant effect. Redox active transition metals have ability to induce and Acknowledgments initiate lipid peroxidation through the production of oxygen radicals, mainly hydroxyl radical, via Fenton’s/Haber-Weiss This work was supported by Spanish Ministerio de Ciencia reactions [63, 406]. Transition metal, including copper [407– e Innovacion´ BFU 2010 20882 and P10-CTS-6494. Mario F. 410], chromium [411, 412], cadmium [413–416], nickel [417, Munoz˜ was supported by Consejeria de Econom´ıa, Innova- 418], vanadium [419–421], manganese [59, 422–424], and cion y Ciencia de la Junta de Andalucia (Spain) postdoctoral iron [59, 407, 425–434] has been utilized to induce lipid fellowship (P10-CTS-6494). peroxidation in vivo mammalian model. References

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